GST Pull Down Assay | Protein-Protein Interaction Using Pull Down Assay |

The glutathione S-transferase (GST) pull-down assay is a relatively easy, straightforward method to identify potential binding partners.

The method is also extensively used to confirm known interactions and to map interaction sites.

The pull-down method relies on the immobilization of a GST fusion protein on glutathione sepharose beads that serve as a solid phase.

The first step requires the expression of the protein of interest as a fusion protein with the GST moiety.

After binding of the GST fusion protein to the glutathione sepharose matrix, the mixture is incubated with whole-cell homogenate or a purified protein or more specifically, the Target proteins tgat are usually lysates of cells either labeled with [35S]methionine or unlabeled, depending on the method used to assay the interaction between the target and the probe. 

Nonbound material is washed off the column, and subsequently the binding complex is eluted.

Upon elution, the mixture is resolved by (SDS-PAGE) and analyzed by Coomassie staining, silver staining, or Western blot....

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